CN113350263B

CN113350263B - Triptolide self-soluble microneedle

Info

Publication number: CN113350263B
Application number: CN202110623619.8A
Authority: CN
Inventors: 张永萍; 陈鑫; 徐剑; 刘耀
Original assignee: Guizhou University of Traditional Chinese Medicine
Current assignee: Guizhou Yangsheng Medical Instrument Co ltd
Priority date: 2021-06-04
Filing date: 2021-06-04
Publication date: 2022-05-17
Anticipated expiration: 2041-06-04
Also published as: CN113350263A

Abstract

The invention discloses a triptolide autolytic microneedle. The tip matrix prescription of the triptolide self-soluble microneedle comprises 24-29 parts by weight of PVP, 7.8-8.3 parts by weight of PVA, 0.45-0.65 part by weight of CMC-Na0.28-0.42 part by weight of triptolide, 40-45 parts by weight of distilled water and 20-23 parts by weight of absolute ethyl alcohol; the backing layer matrix prescription of the triptolide self-soluble microneedle comprises PVP32-37 parts, PVA9-12 parts and distilled water 53-58 parts. The invention has the advantages of good micro-needle formability and mechanical strength, 0.98 percent of RSD value of drug-loading rate, stable and feasible process, the needle point of the drug-loading micro-needle can ensure that most of drugs can quickly reach the body to take effect, and the slow release effect of the micro-needle matrix can improve the medication safety of triptolide, and realizes the treatment of rheumatoid arthritis by reducing the inflammatory infiltration of cells in synovium, reducing the expression of inflammatory factors TNF-alpha, IL-17, PGE2, IgG, IgM and IgA, increasing the expression of OPG, and reducing the trends of the expression of RANKL and the ratio of RANKL/OPG.

Description

Triptolide self-soluble microneedle

Technical Field

The invention belongs to the technical field of autolytic microneedles, and particularly relates to a triptolide autolytic microneedle.

Background

Triptolide (TP), one of the main components of tripterygium glycosides, is both a main active component and a toxic component. Is the main component of the quality control standard of the prior tripterygium wilfordii preparation. Various studies have found that TP has the following properties: (1) the medicament is almost insoluble in pure water, and has the defects of low bioavailability, poor absorption, difficult reaching of therapeutic concentration by blood concentration, difficult preparation due to toxic and side effects caused by high concentration and the like in clinical research; (2) the elimination rate in vivo is high, the nonlinear elimination is realized, and the excrement, urine and bile are mainly excreted; (3) the lethal dose is small, the half-life period is 31 days and 204 days respectively, wherein TP has good stability in chloroform, the degradation of the TP is accelerated by an alkaline medium and a hydrophilic solvent, and the degradation of the TP is influenced by temperature and pH; (5) TP is a neutral compound, does not contain any acidic, basic, easily ionizable groups in the molecule, cannot be efficiently ionized, and cannot generate characteristic fragment ions during collisions, thus having poor mass spectral response. Researches show that TP can inhibit the expression and the generation of IFN gamma in RA synovial fibroblasts, so that Th17 cells are damaged in differentiation, and the effects of immunosuppression and anti-inflammation are achieved; TP can also obviously improve the condition of joint swelling of rats with type II collagen induced arthritis, the mechanism is related to promoting the expression of IL-10 and TGF-beta, increasing the proportion of Treg cells, inhibiting the expression of IL-17, TNF-alpha, VEGF and IFN-gamma, and no obvious hepatotoxicity is generated in a short time. TP and TG are effective medicaments for treating RA, but the clinical application of TP and TG is limited by the factors of high toxicity, low water solubility, unclear treatment targets and the like.

Microneedles (MNs) are a novel physical permeation-promoting technique that can be administered directly through the skin. Has painless penetrating power, can lead water-soluble drugs and macromolecular drugs to penetrate through the horny layer, has good treatment effect, is relatively safe, almost has no damage to the transferred drugs, has stable dosage, low cost and other outstanding performances, and has attracted extensive attention in the scientific and industrial fields in the past decades. However, the self-Dissolving Microneedles (DMNs) among the microneedles are widely used because they can encapsulate a drug and dissolve after being inserted into the skin, and have good biocompatibility. The self-soluble micro-needle also has the function of drug slow release, and the self-soluble micro-needle with different degradation speeds can be manufactured according to the treatment requirement.

At present, the research of triptolide combined with the microneedle is not available.

Disclosure of Invention

The invention aims to provide a triptolide self-soluble microneedle. The invention has the advantages of good micro-needle formability and mechanical strength, 0.98 percent of RSD value of drug-loading rate, stable and feasible process, the needle point of the drug-loading micro-needle can ensure that most of drugs can quickly reach the body to take effect, and the slow release effect of the micro-needle matrix can improve the medication safety of triptolide, and realizes the treatment of rheumatoid arthritis by reducing the inflammatory infiltration of cells in synovium, reducing the expression of inflammatory factors TNF-alpha, IL-17, PGE2, IgG, IgM and IgA, increasing the expression of OPG, and reducing the trends of the expression of RANKL and the ratio of RANKL/OPG.

The invention adopts the following technical scheme to realize the purpose of the invention:

a triptolide autolytic microneedle comprises 24-29 parts by weight of PVP (polyvinyl pyrrolidone), 7.8-8.3 parts by weight of PVA (polyvinyl alcohol), 0.45-0.65 part by weight of CMC-Na0.28-0.42 part by weight of triptolide, 40-45 parts by weight of distilled water and 20-23 parts by weight of absolute ethyl alcohol; the backing layer matrix prescription of the triptolide self-soluble microneedle comprises PVP32-37 parts, PVA9-12 parts and distilled water 53-58 parts.

In the triptolide self-soluble microneedle, the prescription of the tip matrix of the triptolide self-soluble microneedle comprises, by weight, 26-27 parts of PVP, 8-8.1 parts of PVA, 0.5-0.6 part of CMC-Na0.3-0.4 part of triptolide, 42-43 parts of distilled water and 21-22 parts of absolute ethyl alcohol; the backing layer matrix prescription of the triptolide self-soluble microneedle comprises 34-35 parts of PVP (polyvinyl pyrrolidone), 10-11 parts of PVA (polyvinyl alcohol) and 55-56 parts of distilled water.

In the triptolide self-soluble microneedle, the prescription of the tip matrix of the triptolide self-soluble microneedle comprises, by weight, 26.78 parts of PVP (polyvinyl pyrrolidone), 8.03 parts of PVA, 0.54 part of CMC-Na (sodium carboxymethyl cellulose), 0.37 part of triptolide, 42.85 parts of distilled water and 21.42 parts of absolute ethyl alcohol; the backing layer matrix prescription of the triptolide self-soluble microneedle comprises 34.48 parts of PVP (polyvinyl pyrrolidone), 10.34 parts of PVA (polyvinyl alcohol) and 55.17 parts of distilled water.

In the triptolide autolytic microneedle, the triptolide autolytic microneedle is prepared by the following steps:

(1) preparing a needle head: weighing needle tip materials according to a needle tip matrix prescription of triptolide autolytic microneedles, fully dissolving the needle tip materials, uniformly stirring, injecting the needle tip materials into a microneedle mould, centrifuging, removing and collecting a drug-containing matrix solution on the surface of the microneedle mould, and drying the drug-containing matrix solution in a dryer to obtain the microneedle mould with the needle tip containing drugs;

(2) preparation of microneedle backing layer: weighing matrix material according to the formula of the backing layer matrix of triptolide autolytic microneedle, stirring, centrifuging, removing bubbles, injecting microneedle mould containing drug at needle tip, centrifuging, taking out microneedle mould containing drug matrix solution, drying, shaping, and demolding.

(1) preparing a needle head: weighing tip materials according to a tip matrix prescription of the triptolide autolytic microneedle, fully dissolving and uniformly stirring the tip materials, injecting the tip materials into a microneedle mould, centrifuging for 8-12min at 3900-;

(2) preparation of microneedle backing layer: weighing matrix materials according to the backing layer matrix prescription of the triptolide autolytic microneedle, uniformly stirring, centrifuging at 4900-5100r/min for 18-22min to remove bubbles, injecting the microneedle mould containing the drug at the needle point, centrifuging at 3900-4100r/min for 4-6min, taking out the microneedle mould containing the drug-containing matrix liquid, drying, forming, and demoulding.

(1) preparing a needle head: weighing needle tip materials according to a needle tip matrix prescription of the triptolide autolytic microneedle, fully dissolving and uniformly stirring the needle tip materials, injecting the needle tip materials into a microneedle mould, centrifuging the needle tip materials at 4000r/min for 10min, removing and collecting a drug-containing matrix liquid on the surface of the microneedle mould, and putting the microneedle mould into a dryer for drying for 6h to obtain a needle tip drug-containing microneedle mould;

(2) preparation of microneedle backing layer: weighing matrix material according to the backing layer matrix prescription of triptolide autolytic microneedle, stirring uniformly, centrifuging at 5000r/min for 20min to remove bubbles, injecting into microneedle mould containing drug at needle point, centrifuging at 4000r/min for 5min, taking out microneedle mould containing drug matrix solution, drying, shaping, and demoulding.

In the triptolide self-soluble microneedle, the triptolide self-soluble microneedle is used for treating rheumatoid arthritis.

In the triptolide autolytic microneedle, the triptolide autolytic microneedle is used for treating rheumatoid arthritis by reducing inflammatory infiltration of cells in synovium, reducing expression of inflammatory factors TNF-alpha, IL-17, PGE2, IgG, IgM and IgA, increasing expression of OPG, and reducing trends of expression of RANKL and ratio of RANKL/OPG.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention determines the prescription PVP 26.78%, PVA 8.03%, CMC-Na0.54%, triptolide 0.37%, distilled water 42.85% and absolute ethyl alcohol 21.42% of the needle tip matrix of DMNs-TP microneedle; the back lining layer matrix of the microneedle is prepared from 34.48% of PVP, 10.34% of PVA and 55.17% of distilled water. Microneedles were prepared in a two-step centrifugation process proposed herein according to the present invention: preparing a needle head: weighing needle point materials, fully dissolving and uniformly stirring the materials, injecting the materials into a microneedle mould, centrifuging the materials at 4000r/min for 10min, removing and collecting a drug-containing matrix liquid on the surface of the microneedle mould, putting the drug-containing matrix liquid into a dryer, drying the drug-containing matrix liquid for 6h, and taking the drug-containing matrix liquid out for later use; preparation of microneedle backing layer: weighing matrix material, stirring uniformly, centrifuging at 5000r/min for 20min to remove bubbles, injecting into microneedle mould containing drug at needle point, centrifuging at 4000r/min for 5min, taking out microneedle mould containing drug matrix solution, drying, shaping, and demoulding. The obtained microneedle has good formability and mechanical strength, the RSD value of the drug-loading rate is 0.98 percent, and the process is stable and feasible.

2. The invention adopts a two-step centrifugation method to prepare the microneedle, the needle point of the drug-loaded microneedle can ensure that most of the drugs can quickly reach the body to take effect quickly, and the drug safety of triptolide can be improved by the slow release effect of the microneedle matrix.

3. The DMNs-TP microneedle prepared by the invention can realize the treatment of rheumatoid arthritis by reducing inflammatory infiltration of cells in synovium, reducing the expression of inflammatory factors TNF-alpha, IL-17, PGE2, IgG, IgM and IgA, increasing the expression of OPG, and reducing the expression of RANKL and the trend of the ratio of RANKL/OPG.

Drawings

FIG. 1 is a schematic representation of a matrix solution for different bubble removal processes;

FIG. 2 is a graph showing results of a single matrix microneedle array with needles;

FIG. 3 is a graph of the results of PVP versus different ductile material ratios;

FIG. 4 is a representation of microneedles of a substrate in different proportions;

fig. 5 is PVP: PVA (10:3, 3:1) rat skin puncture representation;

FIG. 6 is a representation of microneedles with different water addition amounts;

fig. 7 is a morphology chart of microneedle patches for different drying methods;

fig. 8 is a representation of different plasticizer microneedles;

fig. 9 is a representation of different co-solvent microneedles;

FIG. 10 is a graphical representation of the effect of alcohol addition on microneedles;

figure 11 is a representation of different polyglycoside dosing microneedles;

FIG. 12 is a morphological observation of DMNs-TPs;

FIG. 13 is a skin piercing morphology of DMNs-TPs;

FIG. 14 is a graph showing the change in body weight of rats in each group;

FIG. 15 is a photograph of the paw of each group after rat modeling is completed;

FIG. 16 is a photograph of the paw of each group after the end of the administration;

FIG. 17 is an index chart of organs in each group;

FIG. 18 is synovial H & E staining (x 200) of rats in each group;

FIG. 19 shows H & E staining (X200) of articular cartilage in rats of each group;

FIG. 20 is a standard graph of various inflammatory factors;

FIG. 21 is a Western Blot electrophoresis chart showing the effect of OPG and RANKL proteins in each group.

Wherein, in FIG. 1, A is the result of no treatment, B is the result of ultrasonic treatment, and C is the result of centrifugal treatment;

in FIG. 2, A is the result for 50% PVP, B is the result for 60% PVP, C is the result for 70% PVP, D is the result for 15% PVA, E is the result for 2% HA, F is the result for 1% CMC-Na, G is the result for 15% sodium alginate, H is the result for 10% chondroitin sulfate, I is 15% chondroitin sulfate;

in FIG. 3, A is the result of PVPK30+ carbomer 940, B is the result of PVP + PVA, C is the result of PVP + HAP, D is the result of PVP + sodium alginate, E is the result of PVP + CMC-Na, and F is the result of PVP + Dex-40;

in the scale shown in FIG. 4, A is 1:1, B is 1: 2. c is 1:3, D is 1:4, E is 2:1, F is 2:3, G is 3:1, H is 3: 2. i is 3:4, J is 4:1, K is 4:3, L is 10: 3;

in FIG. 5 to scale, A is 10:3 and B is 3: 1;

in FIG. 6, the amount of water added, A is 6mL, B is 8mL, C is 10mL, D is 12mL, and E is 14 mL;

FIG. 7 shows a drying method, wherein A is room temperature drying, B is oven drying, and C is vacuum drying;

in FIG. 8, plasticizer A is CMC-Na, B is PEG-400, C is glycerol, and D is not added;

FIG. 9 shows co-solvents, A is absolute ethanol, B is PEG-400, C is propylene glycol, and D is glycerol;

in FIG. 10, the amount of co-solvent used, A is 0.2mL, B is 0.4mL, C is 0.6mL, and D is 0.8 mL;

in FIG. 11, the amount of drug added was 6mL for A, 7mL for B, 8mL for C, 9mL for D, and 10mL for E;

in fig. 12, a is a 4 × 10 micrograph and B is a camera panorama;

in FIG. 13, A is a staining pattern (panorama) of methylene blue after the penetration of DMNs-TP, B is a staining pattern (stratum corneum of skin is penetrated, penetration depth is 186.81 μm) of skin penetration H & E of DMNs-TP;

in FIG. 15, A is a blank control group, B is a model group, C is a tripterygium glycosides group, D is a piroxicam group, E is a TG low dose group, F is a TG middle dose group, G is a TG high dose group, H is a TP low dose group, I is a TP middle dose group, J is a TP high dose group;

in FIG. 16, A is a blank control group, B is a model group, C is a tripterygium glycosides group, D is a piroxicam group, E is a TG low dose group, F is a TG middle dose group, G is a TG high dose group, H is a TP low dose group, I is a TP middle dose group, and J is a TP high dose group;

in FIG. 17, A is a cardiac index map, B is a liver index map, C is a spleen index map, D is a lung index map, and E is a kidney index map;

in FIG. 18, A is a blank control group, B is a model group, C is a tripterygium glycosides group, D is a piroxicam group, E is a TG low dose group, F is a TG middle dose group, G is a TG high dose group, H is a TP low dose group, I is a TP middle dose group, J is a TP high dose group;

in FIG. 19, A is a blank control group, B is a model group, C is a tripterygium glycosides group, D is a piroxicam group, E is a TG low dose group, F is a TG middle dose group, G is a TG high dose group, H is a TP low dose group, I is a TP middle dose group, and J is a TP high dose group;

in FIG. 21, A is blank group, B is model group, C is DMNs-TP high dose group, D is piroxicam group, E is DMNs-TG low dose group, F is DMNs-TG medium dose group, G is DMNs-TG high dose group, H is DMNs-TP low dose group, I is Tripterygium wilfordii polyglycoside group, J is DMNs-TP medium dose group.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The experimental procedures used below are, unless otherwise specified, all conventional procedures known in the art and the ingredients or materials used, if not specified, are all commercially available ingredients or materials. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention.

Example 1, a triptolide autolytic microneedle, the tip matrix of which is, by weight, 24 parts of PVP, 7.8 parts of PVA, CMC-na0.45 parts of triptolide, 40 parts of distilled water and 20 parts of absolute ethanol; the backing layer matrix prescription of the triptolide self-soluble microneedle comprises 32 parts of PVP, 9 parts of PVA and 53 parts of distilled water.

The triptolide self-soluble microneedle is prepared by the following steps:

(1) preparing a needle head: weighing a needle tip material according to a needle tip matrix prescription of the triptolide autolytic microneedle, fully dissolving the needle tip material, uniformly stirring, injecting into a microneedle mould, centrifuging at 3900r/min for 8min, removing and collecting a drug-containing matrix liquid on the surface of the microneedle mould, and putting into a dryer for drying for 5h to obtain a microneedle mould with a needle tip containing drug;

(2) preparation of microneedle backing layer: weighing matrix material according to the backing layer matrix prescription of triptolide autolytic microneedle, stirring uniformly, centrifuging at 4900r/min for 18min to remove bubbles, injecting into microneedle mould containing drug at needle point, centrifuging at 3900r/min for 4min, taking out microneedle mould containing drug matrix solution, drying, shaping, and demoulding.

Embodiment 2, a triptolide self-soluble microneedle, the tip matrix prescription of which is, by weight, PVP 29 parts, PVA 8.3 parts, CMC-na0.65 parts, triptolide 0.42 parts, distilled water 45 parts, and absolute ethyl alcohol 23 parts; the backing layer matrix prescription of the triptolide self-soluble microneedle comprises 37 parts of PVP, 12 parts of PVA and 58 parts of distilled water.

The triptolide self-soluble microneedle is prepared by the following steps:

(1) preparing a needle head: weighing tip materials according to a tip matrix prescription of the triptolide autolytic microneedle, fully dissolving and uniformly stirring the materials, injecting the materials into a microneedle mould, centrifuging at 4100r/min for 12min, removing and collecting a drug-containing matrix liquid on the surface of the microneedle mould, and putting the microneedle mould into a dryer for drying for 7h to obtain a microneedle mould with a tip containing drug;

(2) preparation of microneedle backing layer: weighing matrix material according to the backing layer matrix prescription of triptolide autolytic microneedle, stirring well, centrifuging at 5100r/min for 22min to remove bubbles, injecting into microneedle mould containing drug at needle point, centrifuging at 4100r/min for 6min, taking out microneedle mould containing drug matrix solution, drying, shaping, and demoulding.

Example 3, the tip matrix of the triptolide self-soluble microneedle is prepared from, by weight, PVP26 parts, PVA8 parts, CMC-na0.5 part, triptolide 0.3 part, distilled water 42 parts, and absolute ethyl alcohol 21 parts; the backing layer matrix prescription of the triptolide self-soluble microneedle comprises 34 parts of PVP (polyvinyl pyrrolidone), 10 parts of PVA and 55 parts of distilled water.

The triptolide self-soluble microneedle is prepared by the following steps:

(1) preparing a needle head: weighing needle tip materials according to a needle tip matrix prescription of triptolide autolytic microneedles, fully dissolving the needle tip materials, uniformly stirring, injecting into a microneedle mould, centrifuging at 3950r/min for 9min, removing and collecting a drug-containing matrix liquid on the surface of the microneedle mould, and putting into a dryer for drying for 5.5h to obtain a needle tip drug-containing microneedle mould;

(2) preparation of microneedle backing layer: weighing matrix material according to the backing layer matrix prescription of triptolide autolytic microneedle, stirring well, centrifuging at 4950r/min for 19min to remove bubbles, injecting into microneedle mould containing drug at needle point, centrifuging at 3950r/min for 4.5min, taking out microneedle mould containing drug matrix solution, drying, shaping, and demoulding.

Example 4, the tip matrix of the triptolide self-soluble microneedle is prepared from, by weight, PVP27 parts, PVA 8.1 parts, CMC-na0.6 parts, triptolide 0.4 parts, distilled water 43 parts, and absolute ethyl alcohol 22 parts; the backing layer matrix prescription of the triptolide self-soluble microneedle comprises 35 parts of PVP (polyvinyl pyrrolidone), 11 parts of PVA and 56 parts of distilled water.

The triptolide self-soluble microneedle is prepared by the following steps:

(1) preparing a needle head: weighing needle tip materials according to a needle tip matrix prescription of triptolide autolytic microneedles, fully dissolving the needle tip materials, uniformly stirring, injecting the needle tip materials into a microneedle mould, centrifuging at 4050r/min for 11min, removing and collecting a drug-containing matrix liquid on the surface of the microneedle mould, and putting the microneedle mould into a dryer for drying for 6.5h to obtain a needle tip drug-containing microneedle mould;

(2) preparation of microneedle backing layer: weighing matrix material according to the backing layer matrix prescription of triptolide autolytic microneedle, stirring well 5050r/min, centrifuging for 21min to remove bubbles, injecting microneedle mold containing drug at needle point, 4050r/min, centrifuging for 4-6min, taking out microneedle mold containing drug matrix solution, drying, molding, and demolding.

Example 5, the tip matrix of the triptolide self-soluble microneedle is prepared from 26.78 parts by weight of PVP, 8.03 parts by weight of PVA, 0.54 part by weight of CMC-Na, 0.37 part by weight of triptolide, 42.85 parts by weight of distilled water and 21.42 parts by weight of absolute ethyl alcohol; the backing layer matrix prescription of the triptolide self-soluble microneedle comprises 34.48 parts of PVP (polyvinyl pyrrolidone), 10.34 parts of PVA (polyvinyl alcohol) and 55.17 parts of distilled water.

The triptolide self-soluble microneedle is prepared by the following steps:

(1) preparing a needle head: weighing a needle tip material according to a needle tip matrix prescription of the triptolide autolytic microneedle, fully dissolving the needle tip material, uniformly stirring, injecting into a microneedle mould, centrifuging at 4000r/min for 10min, removing and collecting a drug-containing matrix liquid on the surface of the microneedle mould, and drying in a dryer for 6h to obtain a needle tip drug-containing microneedle mould;

Example 6. The triptolide autolytic microneedle prepared in the embodiments 1 to 5 is used for treating rheumatoid arthritis, and the triptolide autolytic microneedle for treating the rheumatoid arthritis is realized by reducing inflammatory infiltration of cells in synovium, reducing the expression of inflammatory factors TNF-alpha, IL-17, PGE2, IgG, IgM and IgA, increasing the expression of OPG, and reducing the trends of the expression of RANKL and the ratio of RANKL/OPG.

In order to obtain the technology and verify the technical effect of the invention, the inventor conducts a large number of related experiments, and partial experiments are recorded as follows:

test example 1. Preparation of DMNs-TP and DMNs-TG

The self-soluble microneedle is a microneedle formed by combining a solid microneedle base part and a soluble needle-shaped structure at the front end of the solid microneedle, and is composed of a soluble or biodegradable substrate, and the substrate of the microneedle can be dissolved after being inserted into skin, so that the microneedle has good biocompatibility. The selection of the matrix material directly influences the preparation of the microneedle, the skin puncture and other properties. The test is designed and prepared for the microneedle, a formula of the self-soluble microneedle substrate is screened primarily through the proportion of materials, the water adding amount and the like, the preparation process is optimized orthogonally through the solvent and the adding amount of the microneedle needle point according to the formability, the mechanical property and the drug loading amount of the microneedle, and the skin puncture effect and the in-vitro dissolution property of the microneedle are investigated.

1 materials of the experiment

1.1 instruments, devices

Name of instrument	Manufacturer of the product
		Microneedle mould (A36 type)	Taizhou micro-core medicine science and technology limited
DZF-6210 vacuum drying oven	Shanghai Qixin scientific instruments Co., Ltd
		H2050R model desk type high speed large capacity freezing centrifuge	HUNAN XIANGYI LABORATORY INSTRUMENTS DEVELOPMENT Co.,Ltd.
101-3AB type electrothermal blowing drying box	TIANJIN TAISITE INSTRUMENT Co.,Ltd.
		TDL-5A type large capacity centrifuge	Shanghai Cheng Jie Instrument and Equipment Co Ltd
SK8210LHC type ultrasonic cleaner	Shanghai Kudos Ultrasonic Instrument Co.,Ltd.

The experimental water is deionized water, and other reagents are analytically pure.

2 preparation method and evaluation index of microneedle

2.1 preparation of microneedles

Verbaan et al studied the effect of different length microneedles penetrating the skin and found that microneedles less than 300 μm were not effective in penetrating the skin. The fluorescence analysis method of Zhang Ming et al compares the effect of the micro-needle with different length penetrating into the abdomen skin of the living mouse, and finds that the height of the better micro-needle is more than 500 μm. In the experiment, a microneedle mould with a needle length of 550 mu m is selected to prepare the microneedle.

In the experiment, two-step centrifugation method is adopted to prepare DMNs-TP and DMNs-TG. Step 1, preparing a micro needle: weighing the matrix material according to a certain proportion, putting the matrix material into a beaker, adding a certain amount of alcohol-containing aqueous solution of a reference substance, fully dissolving and uniformly stirring. Injecting the matrix liquid containing the medicine into a microneedle mould, centrifuging for 10min at 4000r/min (3580 Xg) under the condition of normal temperature, removing and collecting the redundant matrix liquid containing the medicine on the surface of the microneedle mould, drying in a dryer for 6h, and taking out for later use; step 2, preparing a microneedle backing layer: weighing matrix materials, dissolving in deionized water, stirring, centrifuging to remove bubbles, injecting into a microneedle mould, centrifuging at 4000r/min (3580 Xg) for 5min, taking out the microneedle mould containing the drug-containing matrix solution, drying, shaping, and carefully demoulding with curved forceps to obtain the drug-containing autolytic microneedle. And (4) placing the microneedle mould into hot water for ultrasonic cleaning, and then placing the microneedle mould into an air-blast drying oven for drying.

2.2 evaluation of microneedle moldability

In order to examine the formability of the matrix material, four indexes of film forming property, flatness, bubble content and needle content are set for comprehensive evaluation. And the compatibility was examined. The appearance and shape of the film were observed by naked eyes and an upright optical microscope. The specific test items are shown in Table 1-1.

TABLE 1-1 evaluation items of moldability of autolytic microneedle

2.3 evaluation of mechanical Properties of microneedles

The mechanical strength of the microneedle was investigated using aluminum foil puncture and in vitro rat skin puncture experiments. The specific steps are shown in Table 1-2.

TABLE 1-2 microneedle mechanical efficacy observations

3 Pre-prescription Studies with microneedles

The microneedle prepared by the two-step centrifugation method comprises a backing layer and a needle point, and whether a plasticizer and a cosolvent are added into the needle point of the microneedle needs to be examined by considering the problems that the drug loading of the microneedle is low, the mechanical property of the microneedle can be reduced by the drug loading of the needle point and the like. Factors influencing microneedle preparation, such as a matrix solution bubble removing method, a matrix material of the microneedle, the water adding amount of the matrix material and a microneedle drying method, are investigated by experiments, and the type of a microneedle tip plasticizer, the type of a cosolvent and the amount of the cosolvent are determined.

3.1 selection and screening of microneedle matrices

3.1.1 selection of matrix solution out of bubble method

A large amount of bubbles can be generated after the microneedle substrate is dissolved, and the formability of the subsequent microneedle preparation is directly influenced by the existence of the bubbles in the substrate solution. The experiment respectively examines the effects of standing, ultrasonic and centrifugal bubble removal, the time is 20min, the centrifugal revolution is 5000r/min, and the ultrasonic frequency is 53 HZ.

3.1.2 screening of microneedle backing layer Single matrix microneedle preparation

The administration mechanism of the self-soluble micro-needle is that the needle point reaches the dermis layer or the active epidermis layer after the micro-needle penetrates into the skin, and the drug is released along with the melting of the needle body, so that the transdermal administration effect is achieved, and the matrix material is required to have sufficient mechanical strength, solubility and stability after being cured. Matrix materials currently used for microneedle fabrication can be divided into two broad categories, natural materials and synthetic polymer materials. The former mainly includes saccharides and proteins. The latter has the advantages of adjustable structure and performance and relatively low cost. In the experiment, 20%, 30%, 40%, 50%, 60% and 70% PVP solutions are prepared respectively; 10%, 15%, 20%, 25%, 30%, 35%, 40% PVA solution; 1%, 2%, 3% HA solution; 10%, 15%, 20%, 25% chondroitin sulfate solution; 30%, 50%, 70% trehalose solution; 20%, 30% sucrose solution, 20%, 30% alpha-glucan solution; 10% and 15% sodium alginate solution; a single matrix of a 2% CMC-Na solution is poured into a mold to prepare a polymer micro-needle patch, and the comprehensive investigation is carried out by the methods of 'under 2.2' and 'under 2.3'.

3.1.3 screening of microneedle backing layer composite matrix Material microneedle preparation

The PVP substrate is screened out after the single substrate is screened, PVP belongs to a brittle material, the PVP is prepared into the microneedle array, the microneedle array can be fragile, the microneedle prepared by compounding a plurality of materials can make up the defect of the single material, and the effect that 1+1 is larger than 2 is achieved. The preparation process is changed in the preparation process, two or more than two materials are compounded according to different modes, and the microneedles with different structures can be prepared, so that the properties which are not possessed by the microneedles made of single materials are obtained. The current compounding modes mainly comprise four types, namely a uniform type, a lamellar type, a coating type and a particle-coated type. The more uniform coating of the microparticle type and the lamellar type can solve the problem that the loading of the drug may reduce the strength of the material. In the experiment, PVP is mixed with PVA, HA, carbomer 940, sodium alginate, Dex-40 and CMC-Na solution to prepare the microneedle, and the dosage is optimized.

3.1.4 screening of proportion of composite matrix Material for microneedle backing layer

Research shows that 5g of brittle material and 1.5g of tough material are weighed, 10mL of distilled water is added into the brittle material and uniformly dissolved, and then the microneedle with good needle shape and excellent mechanical efficiency can be prepared. The optimal ratio of PVP to PVA is optimized in the experiment. 1:1, 1:2, 1:3, 1:4, 2:1, 2:3, 3:1, 3:2, 3:4, 4:1, 4:2, 4:3 and 10:3 of PVP and PVA are respectively mixed, and the micro-needle is prepared according to the method under item 2.1, wherein the ratio of the matrix material to the added water amount is 13: 20, preparing the composite material microneedle, observing the formability of the composite material microneedle under a microscope, and observing the mechanical efficiency of the microneedle by aluminum foil puncture.

3.2 selection of microneedle backing layer Water addition amount

In the process of preparing the DMNs-TPs, a proper amount of water needs to be added into a matrix material of PVP and PVA for dissolution, and the added amount of water directly influences the mechanical strength of the microneedles. In the experiment, 6mL, 8mL, 10mL, 12mL and 14mL are considered, the proportion of PVP and PVA is 10:3 according to the method under item 2.1, DMNs-TP is prepared, the formability is observed under a camera, and the mechanical strength is observed through aluminum foil puncture.

3.3 selection of drying method

The drying pattern has a large effect on the morphology of the microneedles. In the experiment, three types of drying of a room temperature dryer, drying of a 30 ℃ oven and drying of 30 ℃ in vacuum are investigated, the microneedles are dried in three modes respectively, and the formability scores of the microneedles under '2.2 and 2.3' are used as investigation indexes.

3.4 microneedle tip prescription optimization

3.4.1 selection of microneedle tip plasticizers

When the microneedle is prepared, in order to ensure the mechanical property of the needle point, a plasticizer is added into a needle point substrate to enhance the mechanical efficiency of the microneedle. In the experiment, 0.1g of plasticizer such as CMC-Na, glycerol, PEG-400 and the like is added into the matrix, and the mechanical properties of the microneedle are compared and screened with those of the microneedle without the plasticizer.

3.4.2 choice of cosolvent for microneedle tips

Because the medicines carried by the microneedles in the experiment are all fat-soluble components, in order to better carry the medicines by the microneedles and ensure the medicines to be uniformly carried, a certain amount of cosolvent is added into the matrix material at the needle point in the experiment to increase the dissolution of the medicines. In the experiment, the influence of cosolvents such as absolute ethyl alcohol, glycerol, propylene glycol and the like on the drug dissolution condition and the mechanical strength of the microneedle is examined. Before experiments, 5mg of the methyl-pyrrolidone can be completely dispersed and dissolved in a matrix material after 0.4mL of the cosolvent is added, in order to further increase the drug loading capacity, 7mg of methyl-pyrrolidone (excessive) and 0.4mL of different cosolvents are added into a needle tip matrix material liquid, a microneedle is prepared according to the method under item 2.1, and the drug loading capacity, the drug loading uniformity, the formability and the mechanical performance of the microneedle are observed. Wherein the chromatographic conditions of the A are methanol: water 42:58 (v/v); flow rate: 1.0 mL/min; detection wavelength: 218 nm.

3.4.3 selection of amount of alcohol to be added to the tip of microneedle

The experiments show that the solubility of the methyl cellulose can be improved by adding absolute ethyl alcohol into the needle tip matrix material liquid, but because the matrix material is water-soluble, the addition of the ethyl alcohol can affect the mechanical properties of the micro-needle and the like, and can also affect the drug loading rate of the micro-needle. In the experiment, different alcohol adding amounts of 0.2mL, 0.4mL, 0.6mL and 0.8mL are considered, microneedles are prepared according to the method under item 2.1, and the drug loading amount, the drug loading uniformity and the mechanical property of the microneedles are observed.

3.4.4 orthogonal preferred DMNs-TP microneedle tip prescription

Wherein, the water adding amount, the alcohol adding amount and the drug adding amount are factors, the formability, the mechanical property, the drug loading amount and the drug loading uniformity are evaluation indexes, and the DMNs-TP needle point preparation is preferably selected in the orthogonal test. Wherein the moldability and the mechanical property are objectively scored, and the drug loading rate is 70 when the drug loading rate is 0-50 mu g, 80 when the drug loading rate is 50-100 mu g, 90 when the drug loading rate is 100-150 mu g, and 100 when the drug loading rate is 150-200 mu g; the RSD value score is that when the RSD value is 0-2.5%, the RSD value is 90 points, 2.5-5% is 80 points, 5-7.5% is 70 points, 7.5-10% is 60 points, 10-15% is 50 points, and more than 15 points are 40 points. The weight coefficients are (0.2, 0.2, 0.4, 0.2), the composite score is moldability score × 0.2+ mechanical performance score × 0.2+ drug loading score × 0.4+ RSD value score × 0.2, and the results of the orthogonal table and the orthogonal test table are shown in tables 1 to 3 and 1 to 4.

Tables 1-3 orthogonal factor tables

Tables 1-4 orthogonal test tables

3.4.5 selection of the amount of DMNs-TG added

The result shows that 0.8mL of water and 0.4mL of ethanol can dissolve 7mg of the polyglycoside extract at most, so whether 6mg, 7mg, 8mg, 9mg and 10mg of the polyglycoside extract added into the needle tip material can influence the formability and mechanical efficiency of the microneedle or not is investigated in the experiment, and the drug loading rate and drug loading uniformity of the microneedle are observed at the same time.

4 results of the experiment

4.1 microneedle matrix selection and screening

4.1.1 selection of matrix degassing method

As a result, as shown in FIG. 1, the matrix bubbles were hardly changed after standing for 20 min; after 20min of ultrasonic treatment, about half of bubbles in the matrix are removed; and after 20min of centrifugation, all air bubbles in the matrix are removed. The results show that: the bubble removing effect is the best by centrifugation. The centrifugation time may vary depending on the amount of the prepared matrix solution.

4.1.2 microneedle backing layer results of microneedle fabrication with single matrix material

The results are shown in tables 1-5, and it can be seen that when microneedles are prepared from single substrates with different concentrations, a complete microneedle array and PVP solutions with the concentrations of 50%, 60% and 70% are obtained, and the microneedles have better microneedle shapes; 15% PVA solution; a 2% HA solution; 2% sodium alginate solution; 1% CMC-Na solution; 10% and 15% chondroitin sulfate solution; the results are shown in FIG. 2. From the results, it can be known that PVP is a brittle material, the prepared microneedle patch is prone to fracture, PVA, HA, and the like are relatively tough materials, and a combination of brittle and tough materials is selected in experiments to prepare a composite autolytic microneedle, and in the experiments, the brittle property of the brittle material is improved by adding the tough material into the brittle material. Therefore, PVP is selected as the brittle material in the experiment.

Tables 1-5 results of microneedle fabrication from a single matrix material

Matrix material	Film forming property	Flatness of	Amount of bubbles	Needle content	Mechanical efficiency	Total score
							50% PVP solution	2	3	3	3	Excellent and not easy to bend	11
60% PVP solution	2	3	3	3	Excellent and not easy to bend	11
							70% PVP solution	2	3	3	3	Excellent and not easy to bend	11
15% PVA solution	3	2	3	3	Good, bending	11
							2% HA solution	3	2	2	2	Poor, easily bendable	9
10% chondroitin sulfate solution	3	2	3	3	Good, bending	11
							15% chondroitin sulfate solution	3	2	3	3	Good, bending	11
2% sodium alginate solution	3	2	2	2	Poor, easily bendable	9
							1％CMC-Na	3	2	2	2	Poor, easily bendable	9

4.1.3 microneedle backing layer composite matrix Material results in microneedle fabrication

The results are shown in tables 1-6, and the combination of PVP and PVA to prepare microneedles is excellent in combination, and the results of microneedle arrays and needles are shown in FIG. 3.

TABLE 1-6 evaluation results of PVP and different toughness material ratio formability

Substrate	Film forming property	Flatness of	Amount of bubbles	Needle content	Compatibility	Mechanical efficiency	Total score
								PVP + carbomer 940	3	2	2	3	A	Poor, easily bendable	10
PVP+PVA	3	3	3	3	A	Excellent and not easy to bend	12
								PVP+HA	3	3	3	3	A	Poor, easily bendable	12
PVP and sodium alginate	3	1	3	3	A	Excellent and not easy to bend	10
								PVP+CMC-Na	3	2	3	3	A	Poor, easy to break	11
PVP+Dex-40	2	2	3	3	A	Excellent and not easy to bend	10

4.1.4 screening results of microneedle backing layer composite matrix Material ratios

The results shown in tables 1-7 show that the ratio of PVP to PVA is 3:1 and 10:3, the overall physical properties are better, but according to the puncture of rat skin, the microneedle with the ratio of 10:3 has more holes when puncturing the rat skin than the microneedle with the ratio of 3:1, and the microneedle with the ratio of 10:3 has better puncture effect, so the microneedle with the ratio of 10:3 is selected. The results of the specific puncture conditions are shown in fig. 4 and 5.

TABLE 1-7 results of composite matrix material ratio screening

4.2 microneedle backing layer Water addition results

As a result, as shown in tables 1 to 8, it was found that the DMNs-TP prepared with an added amount of water of 8mL had the best moldability and mechanical strength. The specific characterization results are shown in fig. 6.

Tables 1-8 Performance rating tables for microneedles with different water addition amounts

Adding water volume (mL)	Film forming property	Flatness of	Amount of bubbles	Needle content	Mechanical efficiency	Total score
							6	3	3	3	3	Good and not easy to bend	12
8	3	3	3	3	Excellent and not easy to bend	12
							10	3	3	3	3	Excellent, easy to bend and break	12
12	3	2	3	2	Excellent and easy to break	10
							14	3	2	3	2	Excellent and easy to break	10

4.3 results of drying method selection

Specific scoring results of moldability of different drying modes are shown in tables 1-9, and the morphological graph result is shown in fig. 7, and researches show that after vacuum drying, the microneedle patch is full of bubbles, and the microneedles at the positions of the bubbles are not visible; the microneedle patch appeared shrunken after drying at 30 ℃ constant temperature and the backing layer had a small amount of air bubbles. The microneedle base layer after the dryer is dried at normal temperature is flat, the microneedle array is clear, the number and the needle shape of the microneedles are the same as those of the mold, the needle points are sharp, the lengths of the microneedles are consistent, the shape is good, and the phenomena of needle breakage or needle bending and the like do not occur, so that the dryer has a better drying effect at normal temperature. Drying in an indoor drier in summer, wherein the temperature fluctuates within 30+ -5 deg.C; during preparation in winter, the dryer is placed in a vacuum drying oven, and drying is carried out at a temperature of 30 ℃ without pressurization.

Tables 1-9 tables for microneedle moldability evaluation for different drying methods

Drying method	Film forming property	Flatness of	Amount of bubbles	Needle content	Total score
						Drying at room temperature	3	3	3	3	12
Oven drying	3	2	2	2	9
						Vacuum drying	3	2	1	1	7

4.4 microneedle tip prescription preferred results

4.4.1 selection results for microneedle plasticizers

As can be seen from the results in FIG. 8, the mechanical properties of the microneedles when CMC-Na was added to the microneedle matrix material were stronger than those of the microneedles when none or others were added. The results thus indicate that the addition of CMC-Na to the microneedle tip matrix material can increase the mechanical efficacy of the microneedles.

4.4.2 selection results of Co-solvent for microneedle tips

The results of drug loading and uniformity of the microneedles with the added cosolvent are shown in tables 1-10, and the results of mechanical properties are shown in fig. 1-9. As a result, it was found that the moldability of the microneedles was good after the addition of the co-solvent; when absolute ethyl alcohol is added into the needle point of the microneedle, the drug loading of the microneedle is maximum, the drug loading uniformity of the microneedle is good, the mechanical efficiency of the microneedle has no great influence, and the microneedle can puncture the skin of a rat, so that the absolute ethyl alcohol is selected as a cosolvent in the experiment.

TABLE 1-10 characterization of different co-solvent microneedles

4.4.3 selection results of amount of alcohol added to the tip of microneedle

The results of adding different amounts of absolute ethanol to the microneedle tips are shown in tables 1-11, and the results of the mechanical properties of the microneedles are shown in fig. 10. The result shows that when 0.6mL of ethanol is added in the experiment, the mechanical property of the microneedle tip is not greatly influenced, the drug-loading rate is moderate, and the RSD value is less than 3 percent, thereby meeting the requirement.

TABLE 1-11 characterization of microneedles with varying amounts of added alcohol

4.4.4 Quadrature preferred results

From the range R, the amount of water (a), the amount of alcohol (B), and the amount of drug (C) that most affect the microneedle tip were determined. The results are shown in tables 1-13, and the best extraction process for the needle tip prescription of DMNs-TP is A2B1C3, namely 0.8mL of water, 0.4mL of absolute ethyl alcohol and 7mg of an A reference substance, which is analyzed by the results in tables 1-12.

Tables 1-12 orthogonal test data

From the above, the optimal microneedle tip formula is A2B1C3, that is, the water addition amount is 0.8mL, the alcohol addition amount is 0.4mL, and the drug addition amount is 7 mg.

TABLE 1-13 analysis of variance of orthogonal test data

Factors of the fact	Sum of squares of deviations	Degree of freedom	F ratio	Critical value of F	Significance of
						Adding water volume (mL)	55.336	2	22.031	19.000	*
Amount of alcohol added (mL)	141.556	2	56.359	19.000	*
						Medicine adding amount (mg)	166.889	2	66.445	19.000	*
Error of	2.5117	2

Note: f0.05(1, 2) ═ 19, F values > 19 indicate significant differences.

4.4.5 selection of the amount of DMNs-TG added

The results of the moldability and mechanical effect of the microneedle tips added with different drug amounts are shown in fig. 11, and the drug loading amount and drug loading uniformity of the microneedles are shown in tables 1-14. It can be known that when 8mg of the polyglycoside extract is added to the needle tip of the microneedle, the formability and mechanical efficiency of the microneedle are not greatly affected, and the drug-loading uniformity is good, so that 8mg of the polyglycoside solution is added to the material of the needle tip of the microneedle in the experiment.

TABLE 1-14 characterization of different Polyglycoside dosing microneedles

Determination and verification of 5 DMNs-TP preparation process

5.1 determination of DMNs-TP preparation Process

According to a single-factor experiment, the single microneedle preparation method, the type of a composite matrix, the proportion of the composite matrix, the water adding amount, a matrix solution bubble removing method, a drying method, the type of a cosolvent added at a needle tip, the type of a plasticizer, the use amount of the cosolvent, the drug loading amount, the water adding amount and the like are optimized by an orthogonal test, and finally the preparation process of the DMNs-TP is determined as the preparation of the microneedle by adopting a two-step centrifugation method, wherein the back lining layer is prepared by 34.48 percent of PVP, 10.34 percent of PVA and 55.17 percent of distilled water through the steps of dissolving and then centrifuging, and centrifuging at 5000r/min for 20min to remove the bubble from the matrix solution; the prescription of the microneedle tip is PVP 26.78%, PVA 8.03%, CMC-Na 0.54%, triptolide 0.37%, distilled water 42.85% and absolute ethyl alcohol 21.42%, so that the microneedle tip is fully dissolved. The preparation method comprises placing about 50 μ L of needle point material liquid in PDMS female mold, centrifuging at 4000r/min for 10min in a centrifuge, taking out, scraping off excessive material liquid, drying in a drier for 6 hr, taking out, adding 0.9mL of backing layer material liquid, centrifuging at 4000r/min in a centrifuge for 5min, taking out, drying in a drier, and demolding.

5.2 DMNs-TP microneedle Performance validation

5.2.1 measurement of drug-loading rate and drug-loading uniformity of DMNs-TP

3 parts of DMNs-TP are prepared according to the method, the drug loading capacity is measured, the RSD value of the drug loading capacity of 3 parts of microneedles is measured, whether the drug solution is uniformly distributed or not is observed, the drug loading capacity of the microneedles is 154.46 +/-1.52 mu g, the RSD value is 0.98%, and the results are shown in tables 1-15.

TABLE 1-15 DMNs-TP microneedle drug loading

5.2.2 microneedle formability test

And (5) observing the forming of the micro-needle by naked eyes, and observing whether the needle shape of the micro-needle is clear and complete or not, and taking a picture by using a camera. The prepared microneedles were placed under an upright optical microscope to observe the appearance and shape thereof, and the results are shown in fig. 12.

5.2.3 skin puncture Performance Observation

In order to further determine the puncture effect of the microneedle, the prepared DMNs-TP is quickly pressed into the skin of the unhairing living SD rat and pressed for 1min, then the DMNs are removed, and methylene blue dyeing and H & E dyeing are carried out; the specific steps are carried out according to the method under item 2.3. The results are shown in FIGS. 1 to 13. As a result, it was found that the microneedles were able to penetrate the stratum corneum layer of the skin of rats, and had good mechanical properties.

6 prescription and preparation of autolytic microneedle

6.1 formulation of autolytic microneedles

The needle tip matrix prescription of the DMNs-TP microneedle is determined to be PVP 26.78%, PVA 8.03%, CMC-Na 0.54%, triptolide 0.37%, distilled water 42.85% and absolute ethyl alcohol 21.42%; the needle tip matrix prescription of the DMNs-TG microneedle comprises 26.77% of PVP, 8.03% of PVA, 0.54% of CMC-Na, 0.43% of tripterygium glycosides, 42.82% of distilled water and 21.41% of absolute ethyl alcohol; while the back lining layer matrix of the microneedle is prepared by 34.48 percent of PVP, 10.34 percent of PVA and 55.17 percent of distilled water.

6.2 preparation of autolytic microneedles

Microneedles were prepared according to the "under 2.1" procedure. Preparing a needle head: weighing the needle tip material, fully dissolving and

uniformly stirring, injecting into a microneedle mould, centrifuging at 4000r/min for 10min, removing and collecting the drug-containing matrix liquid on the surface of the microneedle mould, drying in a dryer for 6h, and taking out for later use; preparation of microneedle backing layer: weighing matrix material, stirring uniformly, centrifuging at 5000r/min for 20min to remove bubbles, injecting into microneedle mould containing drug at needle point, centrifuging at 4000r/min for 5min, taking out microneedle mould containing drug matrix solution, drying, shaping, and demoulding.

7 discussion and summary

The formability and the formability of 3 parts of DMNs-TP prepared by the method are measured, and the three parts of microneedles are good in formability and mechanical strength after the drug loading is measured, and the RSD value of the drug loading is 0.98 percent, which shows that the process is stable and feasible.

The microneedle is prepared by a two-step centrifugation method, the needle point of the drug-loaded microneedle can enable most of drugs to quickly reach the body to take effect quickly, and the drug safety of triptolide can be improved due to the slow release effect of the microneedle matrix.

Test example 2. Preliminary pharmacodynamic study of DMNs-TP and DMNs-TG on RA

Rheumatoid Arthritis (RA) is a chronic autoimmune disease characterized primarily by synovial cell proliferation, infiltration of various inflammatory cells, connective tissue proliferation, and destruction of cartilage and bone tissue. Research shows that the expression of TNF-alpha, IL-17, IgG, IgA, IgM and PGE2 is closely related to the pathogenesis of RA. It has also been found that the OPG/RANKL/RANK signaling pathway can reduce the reduction of bone mass, bone erosion and damage around joints, thereby achieving the effect of treating RA.

In the experiment, a pathological model of rheumatoid arthritis is created by a method of carrying out low-temperature isovolumetric full emulsification on bovine II collagen and incomplete Freund's adjuvant and then carrying out subcutaneous injection twice on foot sole. Male SD rats are randomly divided into a blank group, a model group, a piroxicam patch group, a tripterygium glycosides group, a DMNs-TP high, middle and low groups and a DMNs-TG high, middle and low groups. In the experiment, the changes of the weight, the joint index and the swelling degree of the rat during the administration period are observed, meanwhile, the synovial membrane and the cartilage of the joint of each group of rats are subjected to H & E staining to observe the infiltration condition of inflammatory cells, the contents of TNF-alpha, IL-17, IgG, IgA, IgM and PGE2 factors in serum after the administration of each group are detected by ELISA, and the expression of OPG and RANKL in the synovial membrane of the joint of each group of rats is detected by Western Blot. The piroxicam patch and the tripterygium glycosides patch are the marketed formulations with better treatment effect on rheumatoid arthritis, and the piroxicam patch is selected as a positive drug in consideration of the administration mode of the autolytic microneedle; meanwhile, the DMNs-TG and the tripterygium glycosides tablet have the same medicinal effect component, and the tripterygium glycosides tablet is also used as another positive medicine in the experiment.

1 materials of the experiment

1.1 Experimental instruments

Name of instrument	Manufacturer of the product
		Miniature high-speed centrifuge C2500-R-230V	Labnet of America
ICV-450 of electric heating constant temperature incubator	Japanese ASONE
		Flexstation3 multifunctional microplate reader Flexstation3	Molecular Devices
Micropipettor Eppendorf	Beijing Liuyi Instrument Factory
		DYCZ-40 electrotransformation apparatus	Beijing Liuyi Instrument Factory
PH meter PHS-3C	Shanghai Yueping scientific Instrument manufacturing Co., Ltd
		Magnetic stirrer HJ-6A	CHANGZHOU GUOHUA ELECTRIC APPLIANCE Co.,Ltd.
Mu.l ISKANMK3 enzyme labeling instrument	Thermo
		Centrifuge HI650	HUNAN XIANGYI LABORATORY INSTRUMENTS DEVELOPMENT Co.,Ltd.
Pathological microtome RM 2016	German Leica rotary slicer
		JK-6 TYPE OF TISSUE SPREADER	Wuhanjunjie biological tissue spreading and baking machine
Microscope	Olympus BX53 type biological microscope
		Vernier caliper	Shanghai measuring tools and cutting tools factory

1.2 Experimental reagents

1.3 Experimental animals

SD rats (240 ± 20g), lot number: 430726200100342862, respectively; license number: SCXK (Xiang) 2019-.

2 preliminary pharmacodynamic study of autolytic microneedles

2.1 Experimental methods

2.1.1 animal groups

After 100 SD rats are adaptively fed for 1w, the SD rats are randomly divided into blank groups; a model group; tripterygium glycosides autolytic microneedle high, medium and low dose groups; triptolide autolytic microneedle high, medium and low dose groups; piroxicam patch group and Tripterygium glycosides tablet group.

2.1.2 Molding method

Taking the bought collagen solution and the incomplete freon adjuvant out of a refrigerator at 4 ℃ before molding, taking a part of the collagen solution prepared from glacial acetic acid and bovine type II collagen, fully emulsifying the incomplete freon adjuvant in the same volume at low temperature, and dropping a drop of the emulsified collagen solution into a beaker filled with water to form a drop, thereby proving that the collagen solution is fully emulsified. Then, the rats of 9 groups except the normal group are injected with 0.4mL of emulsifier subcutaneously in the left hind foot sole, and the 14 th day of model is injected with 0.1mL of reinforcing model 6d in the right hind foot sole.

2.1.3 preparation of the groups to be administered

Preparing DMNs-TP as a high dose according to a prescription of 'item 6.1' in the first part, wherein the dosage of the medium dose is half of that of the high dose, and the dosage of the low dose is half of that of the medium dose; DMNs-TG was prepared as a high dose according to the first section 'under 6.1' prescription, with the medium dose half the high dose,the low dose is half of the medium dose. The dose of piroxicam patch and Tripterygium glycosides patch is calculated according to the formula of body type coefficient conversion algorithm. The results show that the tripterygium glycosides tablet set is perfused at 5.6mg/kg for 1d/1 time; the piroxicam group is 1 patch per day, namely 4.47 mg/kg. The rest microneedle patch groups have an area of 2.75cm per day²Microneedle patches were administered. Each group was fed water and feed normally daily. After the molding is finished, the administration is started, and the administration time is 28 days.

(D represents species dose (mg/Kg), R represents system coefficient, body surface coefficient of rat is 0.09, body surface coefficient of human is 0.1, W represents body weight, generally human body weight is 60Kg, rat is 0.25 Kg.)

2.1.4 data processing

All data adoption

Showing that SPSS17.0 software is adopted for data processing statistics, single-factor analysis of variance is adopted when measured data accords with normal distribution, an LSD method is adopted when variance is uniform, and Dunnett' sT3 method is adopted for comparison when variance is irregular. P<A difference of 0.05 is statistically significant.

2.2 detection of indicators

2.2.1 rat appearance assessment and changes in body Mass

And observing the glossiness and the mental state of the fur of the rat during the drug administration and the model building, and measuring the mass of the rat body before the model building, the second model building, before the drug administration, 14d of the drug administration and after the drug administration, and well recording.

2.2.2 rat arthritis index score

The hind paw articulation and secondary lesion degree of each group of rats were observed and scored after molding and after administration, respectively, with the scoring criteria shown in table 2-1.

TABLE 2-1 rat Joint index score item Table

2.2.3 Observation of the swelling degree of the foot in rats

Shooting pictures of the paw of the rat before and after drug administration by a camera, and measuring the thickness of the swollen toe of the rat by a vernier caliper to obtain the swollen toe swelling degree; the left hind paw volumes of each group of rats were measured and analyzed before molding, after molding (before dosing), and 14d and 28d dosing, respectively.

2.2.4 rat organ index measurement

Research shows that triptolide and tripterygium glycosides have toxicity to organs. Experiment after administration, rats were sacrificed, and after the heart, liver, spleen, lung, and kidney of each group of rats were removed, fat and fascia were removed, blood was squeezed out, surface liquid was blotted with filter paper and precisely weighed, and the ratio of the weight of the rats to the body (mg/g) was calculated, respectively.

2.2.5H & E staining of synovial membrane and cartilage of rat joints

After the administration of the drug to each group of rats was completed, and after fasting and feeding without water were performed for 24 hours, the synovial joints and the articular cartilage of each group of rats were fixed in 4% paraformaldehyde, and then stained according to the procedure of H & E staining referred to in the literature.

2.2.6ELISA assay of TNF-. alpha.IL-17, IgG, IgA, IgM, PGE2 in rat serum

After the rats are administrated for 28 days, the abdominal aorta is subjected to blood sampling after fasting and water feeding is not prohibited for 24 hours, serum is separated at 3000r/min and 10min, and the content of the indexes in the serum of each group of rats is detected according to the kit method of TNF-alpha, IL-17, IgG, IgA, IgM and PGE2 respectively.

2.2.7Western Blot assay of OPG, RANKL expression in synovial membranes of rat joints

Rats were sacrificed after 28 days of administration and 24h after fasting without water deprivation. The synovial membranes of 3 joints of each group of rats were taken out and placed in an empty EP tube, and then frozen and stored in a refrigerator at-80 ℃. Crushing synovial tissues of various groups of rats which are frozen and preserved at low temperature, adding cell lysate to extract protein, performing electrophoresis (the protein loading amount is 40 mu g), performing constant current electricity transfer to a PVDF membrane, rinsing TBST, adding 5% skimmed milk powder, sealing in the dark for 2h, rinsing for 5 times, adding primary antibody (1/1000 diluent of RANKL and OPG), and keeping the temperature at 4 ℃ overnight. After 3 rinses secondary antibodies (RANKL, 1/50000 dilution of OPG) were added, after 5 rinses TBST ECL was added, and after exposure the film grey values were analyzed with BandScan (GAPDH was chosen as the reference protein).

3 results of the experiment

3.1 appearance and body Mass changes in rats

The results are shown in fig. 14, after the model building, except for the blank group, the rats in each group are listened, the hair is dull, the hind paw is red and swollen, the movement is limited, the hind paw is obviously swollen after 24h, secondary lesion occurs to the lateral paw after 14d, and the paw is red and swollen after 20 d; after administration, the rats in each group were better in spirit, fed normally, and had shiny hair, similar to the normal group. As can be seen from the following body weight change graphs, the body weight gain of each group was significantly reduced after molding except for the blank group, and the body weight gain of each group was increased after the start of administration.

3.2 measurement of rat articular index

The results are shown in the table 2-2, the joint indexes of the groups after molding are very different (P is less than 0.01) compared with the joint indexes of the blank group, and the molding effect of the method is good; compared with the model group, each group has no significant difference, which shows that the molding effect of the method is stable. After the administration is finished, compared with the model group, the piroxicam group, the DMNs-TP high dose group and the DMNs-TG high dose group have extremely obvious difference (P < 0.01); compared with the model group, the difference between the Tripterygium wilfordii multi-glycoside group, the DMNs-TP middle dose group, the DMNs-TP low dose group, the DMNs-TG middle dose group and the DMNs-TG low dose group is obvious (P is less than 0.05); the joint index visual analysis data shows that the therapeutic effects of the piroxicam group, the DMNs-TP high dose group and the DMNs-TG high dose group are better than those of the tripterygium glycosides group, the DMNs-TP medium dose group, the DMNs-TP low dose group, the DMNs-TG medium dose group and the DMNs-TG low dose group; while the effect of the DMNs-TP high dose group was better than that of the DMNs-TG high dose group, it can be seen from the above data that each administration group was the best effect of the DMNs-TP high dose group.

TABLE 2-2 comparison of joint index scores after modeling for each group of rats: (

n is 8, fen)

Group of	Joint index score (before dosing)	Joint index score (after administration)
			Blank control group	0⁴⁾	0⁴⁾
Model set	8.11±0.60²⁾	6.11±0.60²⁾
			Piroxicam group	8.33±1.22²⁾	3.33±0.50²⁾⁴⁾
Tripterygium wilfordii multiglucoside tablet group	7.80±0.42²⁾	4.50±0.53²⁾³⁾
			DMNs-TP high dose group	7.70±0.48²⁾	3.67±0.82²⁾⁴⁾
DMNs-TP Medium dose group	7.89±0.33²⁾	3.71±1.25²⁾³⁾
			DMNs-TP Low dose group	7.80±1.14²⁾	4.00±0.92²⁾³⁾
DMNs-TG high dose group	7.70±1.25²⁾	4.13±0.35²⁾⁴⁾
			DMNs-TG middle dose group	7.70±0.48²⁾	4.50±0.76²⁾³⁾
DMNs-TG Low dose group	7.88±1.13²⁾	4.88±0.64²⁾³⁾

Note: in comparison with the normal group,¹⁾P<0.05；²⁾P<0.01. in comparison with the set of models,³⁾P<0.05；⁴⁾P<0.01。

3.3 rat paw picture and rat swelling degree

The results of different periods of foot swelling degree of each group are shown in tables 2-3, the picture of foot swelling degree of each group of rats after model building is shown in figure 15, and the picture of foot swelling degree of each group after model building is shown in figure 16. As can be seen from tables 2-3, after molding, the swelling degree of feet of each group was significantly increased except for the blank group; compared with the blank group, the joint indexes of the groups are very different (P <0.01), which indicates that the molding effect of the method is stable. After the administration is finished, the difference between each treatment group except the DMNs-TG low-dose group and the model group is very obvious (P is less than 0.01), which indicates that each treatment group except the DMNs-TG low-dose group has treatment effect on RA; the DMNs-TG low dose group has no obvious difference (P is more than 0.05) although the DMNs-TG low dose group has the tendency of improving. Compared with the blank group, except the positive medicine group, the high-dose group and the medium-dose group of the DMNs-TP groups have no obvious difference (P is more than 0.05), and the low-dose group has obvious difference (P is less than 0.01); the difference between the DMNs-TG in the high dose group is not obvious (P is more than 0.05), and the difference between the medium dose group and the low dose group is very obvious (P is less than 0.01); the effect of the DMNs-TP high dose group, the medium dose group and the DMNs-TG high dose group is better than that of the DMNs-TP low dose group and the DMNs-TG medium dose group and the DMNs-TG low dose group, wherein the effect of the DMNs-TP high dose group is best.

TABLE 2-3 swelling degree data of rats at different times for each group: (

n＝8，cm)

3.4 conditions of organ index of rats in each group

The results of the organ index of each group of rats after administration are shown in tables 2 to 4, and the results show that the heart index, the lung index, the spleen index, the kidney index and the liver index of the rats after model creation are all significantly increased, and have significant difference (P <0.01) compared with the blank group. After the administration, the organ indexes of rats in each treatment group are reduced to different degrees, and compared with the model group, the heart index, the liver index, the spleen index and the lung index of the DMNs-TP high-dose group are remarkably reduced (P <0.01) and the kidney index is reduced (P < 0.05); dose group spleen index significantly decreased in DMNs-TP (P < 0.01); a decrease in cardiac index, pulmonary index (P < 0.05); the heart index and the lung index of the DMNs-TP low dose group are reduced (P < 0.05); spleen index of each DMNs-TG dose group is remarkably reduced (P <0.01), and lung index is reduced (P < 0.05); spleen indexes of the tripterygium glycosides tablet group are remarkably reduced (P is less than 0.01); the piroxicam group has reduced heart index, lung index and kidney index (P <0.05), and significantly reduced spleen index (P < 0.01). In conclusion, the DMNs-TP group has the best effect of reducing the organ indexes and has better effect than the tripterygium glycosides group and the piroxicam group, but each DMNs-TG group has better effect of reducing the spleen indexes than other groups and has better effect than the tripterygium glycosides group. The specific organ index trend results are shown in fig. 17.

TABLE 2-4 comparison of organ indices of rats in each group: (

n＝8，mg/g)

3.5H & E staining of rats in each group

The synovial H & E results are shown in FIG. 18, and the synovial cells of the rats in the normal group are arranged regularly and have obvious layers; the model group shows that synovial cells have fuzzy and disorganized veins, fibrous tissue hyperplasia in the lower layer of synovium, inflammatory cell infiltration and connective tissue hyperplasia; the conditions of high DMNs-TP, high DMNs-TG, piroxicam group and tripterygium glycosides group are obviously reduced; the low dose group showed a slight decrease in the above. The synovitis pathology scoring results of each group are shown in tables 2-5, the results show that the normal group has no synovitis, the model group is high synovitis, the DMNs-TP middle and low dose groups and the DMNs-TG middle and low dose groups are medium synovitis, the DMNs-TP high dose group and the DMNs-TG high dose group, and the tripterygium glycosides group and the piroxicam group are mild synovitis; the scores of the model group are obviously increased compared with the normal group, and the groups are obviously reduced after administration, wherein the DMNs-TP high and medium dose groups are reduced most with the DMNs-TG high dose group, the reduction degree of the DMNs-TP high dose group is similar to that of the piroxicam group, and the reduction range of the DMNs-TG high dose group is less approximately same as that of the tripterygium glycosides group; however, compared with the blank, the DMNs-TP high dose group has no difference (P is more than 0.05), and the piroxicam group has difference (P is less than 0.05); the DMNs-TG high dose group and the tripterygium glycosides group have obvious difference (P < 0.01); in conclusion, each dosage group of DMNs-TP and DMNs-TG can inhibit synovium inflammation and angiogenesis of RA rats, and the DMNs-TP high dosage group is the best.

The joint synovium H & E results are shown in FIG. 19, and it can be known from H & E staining that the surface layer structure of the cartilage of the mice in the blank group is complete, the chondrocytes are uniformly distributed, the cartilage matrix is uniformly stained, the tide line is basically smooth, and other obvious changes are not seen; the mouse model group can show that the joint tissue is seriously lost, the disordered part of the cartilage structure is thinned until the cartilage structure is lost, a large amount of cartilage cells are reduced and disappear, and local visible blood cells are dissociated outside; the articular cartilage surfaces of cartilage tissues of other administration groups are more complete than those of the model group, the fissures are reduced, the number of chondrocytes is more than that of the model group, and no blood cells are dissociated outside. The results of the Markin's scores are shown in tables 2-6, compared with the blank group, the Markin's scores outside the DMNs-TP high dose group and the piroxicam group are not obviously different, the Markin's scores of the other administration groups are obviously reduced (P <0.01), compared with the model group, the Markin's scores of the administration groups are obviously reduced (P <0.01), which shows that the administration groups have obvious improvement effect on the articular cartilage of RA rats, and the DMNs-TP high dose group and the piroxicam group are optimal.

Table 2-5 comparison of the knee synovitis scores in the rats in each group (

n is 3, min)

table 2-6 comparison of Markin's scores for rats in each group (

n is 3, min)

Group of	Markin's score
		Blank control group	0±0
Model set	10.67±0.58²⁾⁴⁾
		Tripterygium wilfordii multiglucoside tablet group	2.67±0.58²⁾⁴⁾
Piroxicam group	0.67±0.58⁴⁾
		DMNs-TP high dose group	0.67±0.58⁴⁾
DMNs-TP Medium dose group	2.33±0.58²⁾⁴⁾
		DMNs-TP Low dose group	2.67±0.58²⁾⁴⁾
DMNs-TG high dose group	2.33±0.58²⁾⁴⁾
		DMNs-TG middle dose group	3.00±1.00²⁾⁴⁾
DMNs-TG low dose group	4.33±0.58²⁾⁴⁾

3.6 expression of TNF-. alpha.IL-17, IgG, IgA, IgM, PGE2 in serum

The dose response curves of the standards were plotted against the OD values of the factor standards, and the results are shown in fig. 20. The values of each group were brought to obtain specific doses, and the expression results of TNF-. alpha.IL-17, IgG, IgA, IgM and PGE2 in serum are shown in tables 2-7 and 2-8. The ELISA results of the serum of each group of mice show that the values of inflammatory factors TNF-alpha, IL-17, PGE2, IgG, IgA and IgM in the serum of the model group are higher than those of a blank control group, and the significant difference (P <0.01) shows that the experimental modeling is successful; after the administration is finished, compared with a blank group, PGE2 in the DMNs-TP high dose group has no obvious difference (P is more than 0.05), IgG, IgA and IgM have differences (P is less than 0.05), and other factors have obvious differences (P is less than 0.01); PGE2 in the piroxicam group has no obvious difference (P is more than 0.05), IgG and IgA have difference (P is less than 0.05), and other factors have obvious difference (P is less than 0.01); the PGE2 in the Tripterygium wilfordii polyglycoside group and DMNs-TG high dose group are different (P <0.05), and the other factors are different in significance (P < 0.01); the factors of the other groups are significantly different (P < 0.01). Compared with the model group, the reduction of inflammatory factors except the DMNs-TG low-dose group and the blank group has significant difference (P <0.01), the reduction of TNF-alpha, PGE2 and IgG of the DMNs-TG low-dose group has significant difference (P <0.05), and the reduction of IL-17, IgA and IgM have significant difference (P <0.01), so that the groups have treatment effect on RA. There was a difference in dosage group IgA in DMNs-TP compared to patch group (P < 0.05); the DMNs-TP low dose group has difference of PGE2, IgG and IgA (P <0.05), and has obvious difference of IgM (P < 0.01); the DMNs-TG has differences of IgG, IgA, TNF-alpha and PGE2 (P <0.05) and IgM (P <0.01) in the dose groups; the low-dose groups of DMNs-TG have significant differences of TNF-alpha, PGE2 and IgG (P <0.01) and differences of IgA and IgM (P < 0.05). Compared with the tripterygium glycosides group, the DMNs-TP has significant difference of the IgA dose group (P < 0.01); the DMNs-TP low dose groups have differences of IgA and IgM (P < 0.05); the DMNs-TG low dose groups have significant differences in TNF-alpha, IL-17, PGE2, IgG, IgA and IgM (P < 0.01). In conclusion, the best curative effect is the DMNs-TP high dose group.

TABLE 2-7 expression of TNF-alpha, IL-17, PGE2 in rat serum of each group [ (B)

n is 8, OD value, pg/mL)

Group of	TNF-α	IL-17	PGE2
				Blank control group	46.40±14.87⁴⁾⁶⁾⁸⁾	35.60±9.44⁴⁾⁶⁾⁸⁾	82.24±32.06⁴⁾⁷⁾
Model set	193.39±56.63²⁾⁶⁾⁸⁾	83.55±8.24²⁾⁶⁾⁸⁾	228.15±73.06²⁾⁶⁾⁸⁾
				Tripterygium wilfordii multiglucoside tablet group	115.57±23.04²⁾⁴⁾	56.32±3.50²⁾⁴⁾	126.43±31.70¹⁾⁴⁾
Piroxicam group	93.61±25.59²⁾⁴⁾	51.86±2.53²⁾⁴⁾	114.43±30.73⁴⁾
				DMNs-TP high dose group	95.34±23.53²⁾⁴⁾	45.47±4.81²⁾⁴⁾	123.33±37.90⁴⁾
DMNs-TP Medium dose group	117.91±36.06²⁾⁴⁾	52.41±5.99²⁾⁴⁾	143.21±35.88²⁾⁴⁾
				DMNs-TP Low dose group	123.93±24.39²⁾⁴⁾	57.52±9.86²⁾⁴⁾	159.28±41.20²⁾⁵⁾⁴⁾
DMNs-TG high dose group	111.68±24.36²⁾⁴⁾	52.59±8.95²⁾⁴⁾	126.18±17.57¹⁾⁴⁾
				DMNs-TG middle dose group	135.49±33.52²⁾⁴⁾⁵⁾	56.81±7.47²⁾⁴⁾	159.12±35.26²⁾⁵⁾⁴⁾
DMNs-TG Low dose group	155.12±44.09^2)3)6)8)	62.51±8.47²⁾⁴⁾⁸⁾	181.75±44.04^2)3)6)8)

Note: in comparison with the normal group,¹⁾P<0.05；²⁾P<0.01. in comparison with the set of models,³⁾P<0.05；⁴⁾P<0.01; compared with the piroxicam group,⁵⁾P<0.05；⁶⁾P<0.01; compared with the tripterygium glycosides tablet group,⁷⁾P<0.05；⁸⁾P<0.01。

tables 2 to 8 expression of IgG, IgA and IgM in the serum of rats in each group: (

n is 8, OD value)

Note: in comparison with the normal group,¹⁾P<0.05；²⁾P<0.01. in comparison with the set of models,³⁾P<0.05；⁴⁾P<0.01; in comparison with the piroxicam group,⁵⁾P<0.05；⁶⁾P<0.01; compared with the tripterygium glycosides tablet group,⁷⁾P<0.05；⁸⁾P<0.01。

3.7 expression of OPG, RANKL in synovium

The electrophoresis results are shown in FIG. 21, and the expression results of OPG and RANKL in synovial membranes are shown in tables 2 to 9. The experimental result shows that compared with a blank control group, the protein expression of OPG in the synovial tissue of the ankle joint of the rat in the model group is reduced, the protein expression of RANKL and the ratio of RANKL/OPG are obviously increased (P is less than 0.01), and the model modeling is successful; compared with the model group, each DMNs-TP dosage group and each DMNs-TG dosage group can increase the expression of OPG, reduce the expression of RANKL and the trend of the ratio of RANKL/OPG, and present a certain dose-effect relationship. The high dose group had the best effect, the medium dose group was the second, the low dose group had the tendency to decrease, but the DMNs-TG low dose group had no statistical significance (P > 0.05). As can be seen from the above, DMNs-TG

And each DMNs-TP dosage group has different degrees of treatment effect on RA, and the DMNs-TP high dosage group is optimal.

Tables 2-9 protein expression of OPG, RANKL in synovial membranes of groups of rats and RANKL/OPG ratio

Group of	OPG/GAPDH	RANKL/GAPDH	RANKL/OPG
				Blank control group	0.53±0.11⁴⁾⁸⁾	0.27±0.03⁴⁾⁶⁾⁸⁾	0.53±0.10⁴⁾⁷⁾
Model set	0.24±0.01²⁾⁶⁾⁷⁾	0.84±0.06²⁾⁶⁾⁸⁾	3.54±0.15²⁾⁶⁾⁸⁾
				Tripterygium wilfordii multiglucoside tablet group	0.39±0.04^2)4)5)8)	0.57±0.08²⁾⁴⁾	1.48±0.18¹⁾⁴⁾
Piroxicam group	0.51±0.06⁴⁾⁷⁰	0.47±0.03²⁾⁴⁾	0.94±0.15⁴⁾
				DMNs-TP high dose group	0.56±0.04⁴⁾⁸⁾	0.41±0.06¹⁾⁴⁾⁸⁾	0.74±0.15⁴⁾⁸⁾
DMNs-TP Medium dose group	0.48±0.07⁴⁾⁷⁾	0.49±0.05²⁾⁴⁾	1.05±0.24⁴⁾
				DMNs-TP Low dose group	0.40±0.02¹⁾⁴⁾⁵⁾	0.70±0.11^2)3)6)7)	1.74±0.22¹⁾⁴⁾
DMNs-TG high dose group	0.48±0.07⁴⁾	0.50±0.05²⁾⁴⁾	1.07±0.23⁴⁾
				DMNs-TG middle dose group	0.37±0.02²⁾⁴⁾⁶⁾	0.62±0.04²⁾⁴⁾⁵⁾	1.67±0.19¹⁾⁴⁾
DMNs-TG Low dose group	0.29±0.04²⁾⁶⁾	0.77±0.11²⁾⁶⁾⁸⁾	2.70±0.75²⁾

4 summary and discussion

After the model is made by adopting bovine II collagen and incomplete Freund's adjuvant, the joint swelling degree and the joint index of other groups have no obvious difference compared with the model group except the blank group, which indicates that the model making method is stable. After the model is molded, the foot paw of the model group has obvious swelling, deformity and bone dissolving phenomena compared with the normal group, the symptoms of each group are relieved after the drug administration, and the normal group has no abnormality; h & E staining shows that the synovium and cartilage tissue of the normal group of rats have normal forms, a large amount of inflammatory cell infiltration and connective tissue hyperplasia exist in the synovium tissue of the model group of rats, the structure in the cartilage tissue is disordered, a large amount of cartilage cells are reduced and disappear, and blood cells can be seen locally to be dissociated outside; after administration, the above conditions were reduced in each group, especially in the DMNs-TP high dose group; the organ indexes of rats in each treatment group are reduced to different degrees, the DMNs-TP group has the best effect of reducing the organ indexes and has better effect than the tripterygium glycosides group and the piroxicam group, but each DMNs-TG group has better effect of reducing the spleen indexes than other groups and has better effect than the tripterygium glycosides group. ELISA result analysis shows that the expressions of TNF-alpha, IL-17, PGE2, IgG, IgM and IgA in each treatment group are obviously reduced compared with those in a model group; the immunohistochemical result shows that the expression of OPG in synovial tissues of rats in the model group is obviously reduced, the ratio of RANKL and RANKL/OPG is obviously increased, the expression of OPG in the groups after administration is increased compared with that in the model group, the ratio of RANKL and RANKL/OPG is reduced, and particularly in the DMNs-TP high-dose group.

The experiments show that each dosage group of DMNs-TP and DMNs-TG can improve the inflammatory infiltration of synovial tissue, connective tissue hyperplasia and the like, reduce the levels of TNF-alpha, IL-17, PGE2, IgG, IgM and IgA of inflammatory factors in rat serum, and play a role in treating RA by regulating an OPG/RANKL/RANK signal path. Compared with the piroxicam group, the DMNs-TP high-dose group has almost equivalent curative effects, but the microneedle type drug-carrying agent is low, the patch drug-carrying agent is large, and the cost can be better saved. And the triptolide is prepared into a microneedle dosage form, so that the release of the triptolide can be controlled, the reproductive toxicity of the triptolide is improved, and the bioavailability of TP is better improved. Comparing the DMNs-TG high-dose group with the tripterygium glycosides tablet group, it can be known that the administration dose of the DMNs-TG is lower than that of the tripterygium glycosides intragastric administration group, but the curative effect of the DMNs-TG high-dose group is better than that of the tripterygium glycosides tablet group, so that the microneedle is used as a novel physical transdermal technology, the treatment effect of the microneedle has advantages compared with that of the traditional dosage form, the microneedle can directly act on the administration part, the smaller dose can achieve the same effect as oral administration, and the superiority of the microneedle as the novel physical transdermal technology is further described.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims

1. A triptolide autolytic microneedle is characterized in that: the tip matrix of the triptolide self-soluble microneedle comprises, by weight, PVP24-29 parts, PVA7.8-8.3 parts, CMC-Na0.45-0.65 part, triptolide 0.28-0.42 part, distilled water 40-45 parts and absolute ethyl alcohol 20-23 parts; the backing layer matrix prescription of the triptolide self-soluble microneedle comprises PVP32-37 parts, PVA9-12 parts and distilled water 53-58 parts.

2. The triptolide autolytic microneedle according to claim 1, wherein: the tip matrix of the triptolide self-soluble microneedle comprises, by weight, PVP26-27 parts, PVA8-8.1 parts, CMC-Na0.5-0.6 part, triptolide 0.3-0.4 part, distilled water 42-43 parts and absolute ethyl alcohol 21-22 parts; the backing layer matrix prescription of the triptolide self-soluble microneedle comprises PVP34-35 parts, PVA10-11 parts and distilled water 55-56 parts.

3. The triptolide autolytic microneedle according to claim 2, wherein: the prescription of the tip matrix of the triptolide self-soluble microneedle comprises, by weight, 26.78 parts of PVP (polyvinyl pyrrolidone), 8.03 parts of PVA (polyvinyl alcohol), 0.54 part of CMC-Na0.54 part of triptolide, 42.85 parts of distilled water and 21.42 parts of absolute ethyl alcohol; the backing layer matrix prescription of the triptolide self-soluble microneedle comprises 34.48 parts of PVP (polyvinyl pyrrolidone), 10.34 parts of PVA (polyvinyl alcohol) and 55.17 parts of distilled water.

4. The triptolide autolytic microneedle according to any one of claims 1-3, wherein: the triptolide autolytic microneedle is prepared according to the following steps:

5. The triptolide autolytic microneedle according to claim 4, wherein: the triptolide autolytic microneedle is prepared according to the following steps:

6. The triptolide autolytic microneedle according to claim 5, wherein: the triptolide autolytic microneedle is prepared according to the following steps:

7. The triptolide autolytic microneedle according to any one of claims 1-3, wherein: the triptolide self-soluble microneedle is used for treating rheumatoid arthritis.

8. The triptolide autolytic microneedle according to claim 7, wherein: the triptolide self-soluble microneedle for treating rheumatoid arthritis is realized by reducing inflammatory infiltration of cells in synovium, reducing the expression of inflammatory factors TNF-alpha, IL-17, PGE2, IgG, IgM and IgA, increasing the expression of OPG, and reducing the expression of RANKL and the trend of the ratio of RANKL/OPG.